John P. Horn

Cav1.3 Channel Voltage Dependence, Not Ca2+ Selectivity, Drives Pacemaker Activity and Amplifies Bursts in Nigral Dopamine Neurons

Cav1.3 (α1D) L-type Ca2+ channels have been implicated in substantia nigra (SN) dopamine (DA) neuron pacemaking and vulnerability to Parkinsons disease. These effects may arise from the depolarizing current and cytoplasmic Ca2+ elevation produced by Cav1.3 channels at subthreshold membrane potentials. However, the assumption that the Ca2+ selectivity of Cav1.3 channels is essential has not been tested. In this study the properties of SN DA neuron L-type Ca2+ channels responsible for driving pacemaker activity in juvenile rat brain slices were probed by replacing native channels blocked with the dihydropyridine nimodipine with virtual channels generated by dynamic clamp. Surprisingly, virtual L-type channels that mimic native and recombinant Cav1.3 channels supported pacemaker activity even though dynamic clamp currents are not carried by Ca2+. This effect is specific because pacemaker activity could not be restored by tonic current injection, virtual nonselective leak channels or virtual NMDA receptors, which share with L-type channels a negative slope conductance region in their current–voltage (I–V) curve. Altering virtual channels showed that the production of pacemaker activity depended on the characteristic voltage dependence of DA neuron L-type channels, while activation kinetics and reversal potential were not critical parameters. Virtual L-type channels also supported slow oscillatory potentials and enhanced firing rate during evoked bursts. Thus, Cav1.3 channel voltage dependence, rather than Ca2+ selectivity, drives pacemaker activity and amplifies bursts in SN DA neurons.

D2 Autoreceptors Chronically Enhance Dopamine Neuron Pacemaker Activity

Activation of D2 autoreceptors on midbrain dopamine neurons has been shown previously to acutely open K+ channels to inhibit intrinsically generated pacemaker activity. Here we report that D2 autoreceptors act chronically to produce an opposite action: to increase the speed and regularity of repetitive action potential firing. Voltage-, current-, and dynamic-clamp experiments, using conventional whole-cell and perforated patch-clamp recording, with cultured rat midbrain dopamine neurons show that a change in the number of functional A-type K+ channels alters firing rate and susceptibility to irregularity produced by other channels. cAMP and protein kinase A mediate the long-term action of D2 receptors in a manner that counters the short-term effect of this signaling pathway on K+ channel gating. We conclude that D2 autoreceptors, in addition to mediating acute negative feedback, are responsible for long-term enhancement of the rate and fidelity of dopamine neuron pacemaker activity.

Double labeling of the paravertebral sympathetic C system in the bullfrog with antisera to LHRH and NPY.

The specificity of synaptic contacts between pre- and postganglionic cells in the sympathetic C system has been examined by immunocytochemical localization of two neuropeptides. Sections of bullfrog paravertebral sympathetic ganglia were stained with antibodies to luteinizing hormone releasing hormone (LHRH) and neuropeptide Y (NPY). Preganglionic synaptic boutons containing LHRH immunoreactivity were found to make contact with a subpopulation of postganglionic cell bodies and with some clusters of small intensely fluorescent (SIF) cells. In ganglia 9 and 10, 95.8% of the neurons contacted by LHRH-containing boutons were also positive for NPY-like immunoreactivity and conversely, 99.3% of the neurons that contained NPY-like immunoreactivity were contacted by LHRH-containing boutons. Qualitatively similar results were found in most other paravertebral ganglia. These observations support the conclusions that preganglionic C axons selectively innervate C-type ganglion cells and that virtually all C-type ganglion cells and some SIF cells receive a direct LHRH input. Moreover, they suggest that a pattern of specific connections between two sets of peptidergic neurons is expressed throughout most of the paravertebral sympathetic chain of the bullfrog.

Pacemaker Rate and Depolarization Block in Nigral Dopamine Neurons: A Somatic Sodium Channel Balancing Act

Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density, and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency.

Presynaptic muscarinic inhibition in bullfrog sympathetic ganglia.

1. Muscarinic modulation of nicotinic transmission was studied in bullfrog sympathetic ganglia by recording synaptic currents from B and C neurones. 2. Bath‐applied muscarine reduced the amplitude of EPSCs recorded at < 0.2 Hz from B neurones by up to 57%. The action was reversible, showed no apparent desensitization, and had an EC50 of 102 nM. Muscarine had no effect on EPSCs in C neurones. 3. Currents evoked by ionophoretic application of ACh to B neurones were unchanged by muscarine. Muscarine increased the coefficient of variation (c.v.) of EPSC amplitude. The effect upon the ratio of c.v.2control to c.v.2muscarine was proportional to the change in mean EPSC amplitude. 4. Activation of muscarinic receptors by ACh from nerve terminals was observed by comparing trains of EPSCs in normal Ringer solution and atropine. Inhibition of EPSC amplitude by 15‐40% was seen as frequency was increased from 1 to 5 Hz. The minimal latency for onset of inhibition was approximately 2 s. Stimulation at 20 Hz did not produce inhibition. 5. The results indicate that presynaptic muscarinic receptors are selectively expressed by a functional subclass of preganglionic sympathetic nerve terminals. Physiological activation of the receptors occurs during repetitive activity. The extent of autoreceptor‐mediated inhibition varies as a biphasic function of stimulus frequency.

Role of ganglionic cotransmission in sympathetic control of the isolated bullfrog aorta.

1. The relation between preganglionic activity and arterial tone was studied in preparations of bullfrog lumbar sympathetic ganglia 7‐10 and the dorsal aorta. 2. Two or more stimuli evoked contractions when applied to the preganglionic C, but not the B pathway. Contractions were blocked when transmission in ganglia 9 and 10 was disrupted by cutting the sympathetic chain or adding (+)‐tubocurarine. Contractions were antagonized by postganglionic action of guanethidine, but not by phentolamine or suramin. 3. Aortic responses to short trains (10‐100 stimuli) were half‐maximal at 0.3‐0.5 Hz, saturated near 1 Hz and had a minimum latency of 8.9 s. By contrast, responses to 300 stimuli were half‐maximal at 1 Hz and became 2.5‐fold larger at 10 Hz. 4. Exogenous luteinizing hormone releasing hormone (LHRH) potentiated preganglionically evoked contractions. Endogenous LHRH mediated contractions evoked by 10 Hz stimulation in (+)‐tubocurarine. These responses had a longer latency than in normal Ringer solution and were blocked by [D‐pGlu1, D‐Phe2, D‐Trp3.6]‐LHRH. The LHRH antagonist did not alter contractions evoked by continuous stimulation in normal Ringer solution or by bursts of stimuli in hexamethonium. 5. Exogenous neuropeptide Y (NPY) potentiated neurogenic contractions and responses to adrenaline. Benextramine blocked contractions produced by nerve stimulation, adrenaline and NPY, but not ATP. 6. The results show that contractions of the isolated aorta are tuned to physiological frequencies of activity in sympathetic C neurones. Peptidergic cotransmission in the ganglia can increase arterial tension, but not during synchronous activation of primary nicotinic synapses. It is suggested that the physiological role of LHRH arises from interactions with subthreshold nicotinic EPSPs and that postganglionic release of NPY shifts frequency tuning of the circuit during prolonged activity.

Dopamine Neuron Responses Depend Exponentially on Pacemaker Interval

Midbrain dopamine neuron activity results from the integration of the responses to metabo- and ionotropic receptors with the postsynaptic excitability of these intrinsic pacemakers. Interestingly, intrinsic pacemaker rate varies greatly between individual dopamine neurons and is subject to short- and long-term regulation. Here responses of substantia nigra dopamine neurons to defined dynamic-clamp stimuli were measured to quantify the impact of cell-to-cell variation in intrinsic pacemaker rate. Then this approach was repeated in single dopamine neurons in which pacemaker rate was altered by activation of muscarinic receptors or current injection. These experiments revealed a dramatic exponential dependence on pacemaker interval for the responses to voltage-gated A-type K+ channels, voltage-independent cation channels and ionotropic synapses. Likewise, responses to native metabotropic (GABAb and mGluR1) inhibitory synapses depended steeply on pacemaker interval. These results show that observed variations in dopamine neuron pacemaker rate are functionally significant because they produce a >10-fold difference in responses to diverse stimuli. Both the magnitude and the mathematical form of the relationship between pacemaker interval and responses were not previously anticipated.

Functional characterization of ether‐à‐go‐go‐related gene potassium channels in midbrain dopamine neurons – implications for a role in depolarization block

Bursting activity by midbrain dopamine neurons reflects the complex interplay between their intrinsic pacemaker activity and synaptic inputs. Although the precise mechanism responsible for the generation and modulation of bursting in vivo has yet to be established, several ion channels have been implicated in the process. Previous studies with nonselective blockers suggested that ether‐à‐go‐go‐related gene (ERG) K+ channels are functionally significant. Here, electrophysiology with selective chemical and peptide ERG channel blockers (E‐4031 and rBeKm‐1) and computational methods were used to define the contribution made by ERG channels to the firing properties of midbrain dopamine neurons in vivo and in vitro. Selective ERG channel blockade increased the frequency of spontaneous activity as well as the response to depolarizing current pulses without altering spike frequency adaptation. ERG channel block also accelerated entry into depolarization inactivation during bursts elicited by virtual NMDA receptors generated with the dynamic clamp, and significantly prolonged the duration of the sustained depolarization inactivation that followed pharmacologically evoked bursts. In vivo, somatic ERG blockade was associated with an increase in bursting activity attributed to a reduction in doublet firing. Taken together, these results show that dopamine neuron ERG K+ channels play a prominent role in limiting excitability and in minimizing depolarization inactivation. As the therapeutic actions of antipsychotic drugs are associated with depolarization inactivation of dopamine neurons and blockade of cardiac ERG channels is a prominent side effect of these drugs, ERG channels in the central nervous system may represent a novel target for antipsychotic drug development.

In vitro relation between preganglionic sympathetic stimulation and activity of cutaneous glands in the bullfrog.

1. Activation of cutaneous glands was studied by measuring changes in transepithelial potentiation (TEP) after pre‐ and postganglionic sympathetic stimulation in the bullfrog, Rana catesbeiana. 2. In normal Ringer solution, TEP was 20‐90 mV with the basolateral (inside) surface positive. Single shocks to the preganglionic B pathway decreased TEP by up to 3 mV. Cutaneous depolarizations had a latency of 1.2 s, a rise time of 2.5 s, and decayed with an exponential time constant of 15 s. Similar depolarizations were evoked by postganglionic stimulation. 3. Cutaneous depolarizations summed during repetitive stimulation and > 0.05 Hz. For trains of three stimuli, peak amplitude increased with frequency and saturated at 2 Hz. In some preparations, longer trains evoked polyphasic changes in TEP. Preganglionically evoked cutaneous responses were abolished by (+)‐tubocurarine. Postganglionically evoked cutaneous depolarizations were antagonized by phentolamine, but not propranolol. 4. Repetitive preganglionic stimulation of the C pathway (> 100 at 20 Hz) evoked little change in TEP and did not modulate depolarizations evoked through the B pathway. In nicotine, peptidergic cotransmission was enhanced in the ganglia, and repetitive C pathway stimulation evoked cutaneous depolarizations whose time course mirrored that of the postganglionic peptidergic after‐discharge. The after‐discharge and associated cutaneous depolarization were blocked by a luteinizing hormone‐releasing hormone antagonist. 5. The results show cutaneous glands are selectively innervated by B neurones and respond to low levels of neural activity. Asynchronous postganglionic firing mediated by peptidergic cotransmission can provide a basis for heterosynaptic interactions between the B and C pathways.

Regulation of Neuronal Proapoptotic Potassium Currents by the Hepatitis C Virus Nonstructural Protein 5A

Apoptosis-enabling neuronal potassium efflux is mediated by an enhancement of K+ currents. In cortical neurons, increased currents are triggered by dual phosphorylation of Kv2.1 by Src and p38 at channel residues Y124 and S800. It was recently shown that a K+ current surge is also present in hepatocytes undergoing apoptosis, and that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) could inhibit Kv2.1-mediated currents and block cell death. Here, we show that NS5A1b (from HCV genotype 1b) expression in rat neurons depresses delayed rectifier potassium currents, limits the magnitude of the K+ current surge following exposure to activated microglia, and is neuroprotective. In a non-neuronal recombinant expression system, cells expressing Kv2.1 mutated at residue Y124, but not S800 mutants, are insensitive to NS5A1b-mediated current inhibition. Accordingly, NS5A1b coexpression prevents phosphorylation of wild-type Kv2.1 by Src at Y124, but is unable to inhibit p38 phosphorylation of the channel at S800. The actions of the viral protein are genotype-selective, as NS5A1a does not depress neuronal potassium currents nor inhibit Src phosphorylation of Kv2.1. Our results indicate that NS5A1b limits K+ currents following injury, leading to increased neuronal viability. NS5A1b may thus serve as a model for a new generation of neuroprotective agents.